The biosynthesis of pyrimidines requires six enzymatic activities. In mammals, the last two enzymes in this phatway (OPRTase and OMPdecase) are tightly coupled and may exist on a single multi-functional polypeptide. In this proposal, experiments are described using DNA-mediated gene transfer in concert with recombinant DNA technology to isolate the gene coding for the last two enzymes in the pyrimidine biosynthetic pathway. The availability of this gene will: 1) resolve the question as to whether these enzymatic functions are coded for by two discrete genes or a single multi-functional polypeptide (if the latter is true, then the process of gene fusion can be traced in evolutionary time); 2) permit the study of the molecular events associated with the control of synthesis of these enzymatic activities as part of a larger gene battery; and 3) facilitate an analysis of the molecular nature of mutation in somatic cells and in clinical material from patients with defects in this pathway.